Barry P. Young

Phosphatidic acid is a pH biosensor that links membrane biogenesis to metabolism

Intracellular pH and Lipid Metabolism Intracellular pH regulates metabolism by poorly understood mechanisms, but biosensors are likely to be important in this process. Young et al. (p. 1085) took a systems-biology approach in yeast to identify in excess of 200 genes that regulate phospholipid metabolism. They found that the signaling lipid, phosphatidic acid, appeared to act as a cytosolic biosensor via the pH-dependent binding of protein effectors to phosphatidic acid. This pH-dependent mechanism directly affects gene expression and is involved in a pathway in which nutrient availability regulates phospholipid metabolism to control production of membranes. Lipid signaling in yeast is regulated by intracellular pH. Recognition of lipids by proteins is important for their targeting and activation in many signaling pathways, but the mechanisms that regulate such interactions are largely unknown. Here, we found that binding of proteins to the ubiquitous signaling lipid phosphatidic acid (PA) depended on intracellular pH and the protonation state of its phosphate headgroup. In yeast, a rapid decrease in intracellular pH in response to glucose starvation regulated binding of PA to a transcription factor, Opi1, that coordinately repressed phospholipid metabolic genes. This enabled coupling of membrane biogenesis to nutrient availability.

A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria

Tethering of the endoplasmic reticulum to mitochondria by a conserved endoplasmic reticulum complex is needed for the transfer of phospholipids between these organelles.

ER-shaping proteins facilitate lipid exchange between the ER and mitochondria in S. cerevisiae

Summary The endoplasmic reticulum (ER) forms a network of sheets and tubules that extends throughout the cell. Proteins required to maintain this complex structure include the reticulons, reticulon-like proteins, and dynamin-like GTPases called atlastins in mammals and Sey1p in Saccharomyces cerevisiae. Yeast cells missing these proteins have abnormal ER structure, particularly defects in the formation of ER tubules, but grow about as well as wild-type cells. We screened for mutations that cause cells that have defects in maintaining ER tubules to grow poorly. Among the genes we found were members of the ER mitochondria encounter structure (ERMES) complex that tethers the ER and mitochondria. Close contacts between the ER and mitochondria are thought to be sites where lipids are moved from the ER to mitochondria, a process that is required for mitochondrial membrane biogenesis. We show that ER to mitochondria phospholipid transfer slows significantly in cells missing both ER-shaping proteins and the ERMES complex. These cells also have altered steady-state levels of phospholipids. We found that the defect in ER to mitochondria phospholipid transfer in a strain missing ER-shaping proteins and a component of the ERMES complex was corrected by expression of a protein that artificially tethers the ER and mitochondria. Our findings indicate that ER-shaping proteins play a role in maintaining functional contacts between the ER and mitochondria and suggest that the shape of the ER at ER–mitochondria contact sites affects lipid exchange between these organelles.

Inheritance of cortical ER in yeast is required for normal septin organization

How cells monitor the distribution of organelles is largely unknown. In budding yeast, the largest subdomain of the endoplasmic reticulum (ER) is a network of cortical ER (cER) that adheres to the plasma membrane. Delivery of cER from mother cells to buds, which is termed cER inheritance, occurs as an orderly process early in budding. We find that cER inheritance is defective in cells lacking Scs2, a yeast homologue of the integral ER membrane protein VAP (vesicle-associated membrane protein–associated protein) conserved in all eukaryotes. Scs2 and human VAP both target yeast bud tips, suggesting a conserved action of VAP in attaching ER to sites of polarized growth. In addition, the loss of either Scs2 or Ice2 (another protein involved in cER inheritance) perturbs septin assembly at the bud neck. This perturbation leads to a delay in the transition through G2, activating the Saccharomyces wee1 kinase (Swe1) and the morphogenesis checkpoint. Thus, we identify a mechanism involved in sensing the distribution of ER.

Plasma membrane—endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis

Synthesis of phospholipids, sterols and sphingolipids is thought to occur at contact sites between the endoplasmic reticulum (ER) and other organelles because many lipid‐synthesizing enzymes are enriched in these contacts. In only a few cases have the enzymes been localized to contacts in vivo and in no instances have the contacts been demonstrated to be required for enzyme function. Here, we show that plasma membrane (PM)—ER contact sites in yeast are required for phosphatidylcholine synthesis and regulate the activity of the phosphatidylethanolamine N‐methyltransferase enzyme, Opi3. Opi3 activity requires Osh3, which localizes to PM–ER contacts where it might facilitate in trans catalysis by Opi3. Thus, membrane contact sites provide a structural mechanism to regulate lipid synthesis.

Polarization of the Endoplasmic Reticulum by ER-Septin Tethering

Polarization of the plasma membrane (PM) into domains is an important mechanism to compartmentalize cellular activities and to establish cell polarity. Polarization requires formation of diffusion barriers that prevent mixing of proteins between domains. Recent studies have uncovered that the endoplasmic reticulum (ER) of budding yeast and neurons is polarized by diffusion barriers, which in neurons controls glutamate signaling in dendritic spines. The molecular identity of these barriers is currently unknown. Here, we show that a direct interaction between the ER protein Scs2 and the septin Shs1 creates the ER diffusion barrier in yeast. Barrier formation requires Epo1, a novel ER-associated subunit of the polarisome that interacts with Scs2 and Shs1. ER-septin tethering polarizes the ER into separate mother and bud domains, one function of which is to position the spindle in the mother until M phase by confining the spindle capture protein Num1 to the mother ER.

Balony:a software package for analysis of data generated by synthetic genetic array experiments

BackgroundSynthetic Genetic Array (SGA) analysis is a procedure which has been developed to allow the systematic examination of large numbers of double mutants in the yeast Saccharomyces cerevisiae. The aim of these experiments is to identify genetic interactions between pairs of genes. These experiments generate a number of images of ordered arrays of yeast colonies which must be analyzed in order to quantify the extent of the genetic interactions. We have designed software that is able to analyze virtually any image of regularly arrayed colonies and allows the user significant flexibility over the analysis procedure.Results“Balony” is freely available software which enables the extraction of quantitative data from array-based genetic screens. The program follows a multi-step process, beginning with the optional preparation of plate images from single or composite images. Next, the colonies are identified on a plate and the pixel area of each is measured. This is followed by a scoring module which normalizes data and pairs control and experimental data files. The final step is analysis of the scored data, where the strength and reproducibility of genetic interactions can be visualized and cross-referenced with information on each gene to provide biological insights into the results of the screen.ConclusionsAnalysis of SGA screens with Balony can be either automated or highly interactive, enabling the user to customize the process to their specific needs. Quantitative data can be extracted at each stage for external analysis if required. Beyond SGA, this software can be used for analyzing many types of plate-based high-throughput screens.

Critical Determinants for Chromatin Binding by Saccharomyces cerevisiae Yng1 Exist Outside of the Plant Homeodomain Finger

The temporal and spatial regulation of histone post-translational modifications is essential for proper chromatin structure and function. The Saccharomyces cerevisiae NuA3 histone acetyltransferase complex modifies the amino-terminal tail of histone H3, but how NuA3 is targeted to specific regions of the genome is not fully understood. Yng1, a subunit of NuA3 and a member of the Inhibitor of Growth (ING) protein family, is required for the interaction of NuA3 with chromatin. This protein contains a C-terminal plant homeodomain (PHD) finger that specifically interacts with lysine 4-trimethylated histone H3 (H3K4me3) in vitro. This initially suggested that NuA3 is targeted to regions bearing the H3K4me3 mark; however, deletion of the Yng1 PHD finger does not disrupt the interaction of NuA3 with chromatin or result in a phenotype consistent with loss of NuA3 function in vivo. In this study, we uncovered the molecular basis for the discrepancies in these data. We present both genetic and biochemical evidence that full-length Yng1 has two independent histone-binding motifs: an amino-terminal motif that binds unmodified H3 tails and a carboxyl-terminal PHD finger that specifically recognizes H3K4me3. Although these motifs can bind histones independently, together they increase the apparent association of Yng1 for the H3 tail.

Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility.

Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations.

Systematic identification of genes involved in metabolic acid stress resistance in yeast and their potential as cancer targets.

ABSTRACT A hallmark of all primary and metastatic tumours is their high rate of glucose uptake and glycolysis. A consequence of the glycolytic phenotype is the accumulation of metabolic acid; hence, tumour cells experience considerable intracellular acid stress. To compensate, tumour cells upregulate acid pumps, which expel the metabolic acid into the surrounding tumour environment, resulting in alkalization of intracellular pH and acidification of the tumour microenvironment. Nevertheless, we have only a limited understanding of the consequences of altered intracellular pH on cell physiology, or of the genes and pathways that respond to metabolic acid stress. We have used yeast as a genetic model for metabolic acid stress with the rationale that the metabolic changes that occur in cancer that lead to intracellular acid stress are likely fundamental. Using a quantitative systems biology approach we identified 129 genes required for optimal growth under conditions of metabolic acid stress. We identified six highly conserved protein complexes with functions related to oxidative phosphorylation (mitochondrial respiratory chain complex III and IV), mitochondrial tRNA biosynthesis [glutamyl-tRNA(Gln) amidotransferase complex], histone methylation (Set1C–COMPASS), lysosome biogenesis (AP-3 adapter complex), and mRNA processing and P-body formation (PAN complex). We tested roles for two of these, AP-3 adapter complex and PAN deadenylase complex, in resistance to acid stress using a myeloid leukaemia-derived human cell line that we determined to be acid stress resistant. Loss of either complex inhibited growth of Hap1 cells at neutral pH and caused sensitivity to acid stress, indicating that AP-3 and PAN complexes are promising new targets in the treatment of cancer. Additionally, our data suggests that tumours may be genetically sensitized to acid stress and hence susceptible to acid stress-directed therapies, as many tumours accumulate mutations in mitochondrial respiratory chain complexes required for their proliferation. Summary: Altered metabolism in tumours creates metabolic acid stress in tumour cells, which is a target for chemotherapeutics. We identify six new complexes with roles in resistance to metabolic acid stress.